The tibial fracture-pin model is a clinically relevant model of orthopedic trauma comprising a unilateral open tibial fracture with intramedullary nail internal fixation and simultaneous injury to the tibialis anterior muscle. Thermal sensitivity in this model can be measured using a 45 s hot plate paradigm.
The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue damage that manifests with this type of injury in humans. This model was developed because previous models of orthopedic trauma did not include simultaneous injury to multiple tissue types (bone, muscle, nerves) and were not truly representative of human complex orthopedic trauma. The authors therefore modified previous models of orthopedic trauma and developed the tibial fracture-pin model. This modified fracture model consists of a unilateral open tibial fracture with intramedullary nail (IMN) internal fixation and simultaneous tibialis anterior (TA) muscle injury, resulting in mechanical allodynia that lasts up to 5 weeks post injury. This series of protocols outlines the detailed steps to perform the clinically relevant orthopedic trauma tibial fracture-pin model, followed by a modified hot plate assay to examine nociceptive changes after orthopedic injury. Taken together, these detailed, reproducible protocols will allow pain researchers to expand their toolkit for studying orthopedic trauma-induced pain.
Orthopedic trauma accounts for 25% of all injuries sustained by nearly 500 million people each year worldwide1,2,3. Orthopedic trauma can be associated with complex muscle, bone, nerve, and connective tissue damage, necessitating hospitalization and surgery to ensure adequate recovery3,4. Acute and chronic pain after orthopedic trauma can result in significant physical, psychological, and financial burdens that affect a patient's quality of life1,4. Additionally, orthopedic surgery to stabilize and fix fractures is also associated with severe acute and chronic post-surgical pain5,6,7,8,9.
The mechanisms underlying acute and chronic trauma-related pain need to be better understood to develop better treatments. To achieve this, reliable, reproducible, and clinically relevant preclinical models are required. Since most animal models of orthopedic trauma did not involve simultaneous injury to multiple tissue types (bone, muscle, nerves), they were not truly representative of human complex orthopedic trauma, for example, trauma after falls, motor vehicle crashes, or war-related injuries10,11. Therefore, we developed the tibial fracture-pin mouse model to examine the major manifestations of such injury, including bone and muscle tissue damage and acute and chronic pain11. The tibial fracture-pin model consists of a unilateral open tibial fracture with IMN internal fixation and simultaneous TA muscle injury. Histological sections of the TA show injury to the muscle in which dense fibrosis develops with associated loss of large, mature muscle fibers as early as 2 weeks post injury. Moreover, the fracture callus is apparent on microcomputer tomography (microCT) 4 weeks post injury and continues to undergo remodeling11.
Various reflexive and nonreflexive behavior assays can be used to evaluate the sensory and affective components of pain in the tibial fracture-pin model. For example, one can use the Von Frey filaments to demonstrate mechanical hypersensitivity in this model. In fact, mice develop long-lasting mechanical hypersensitivity in the ipsilateral hind paw after tibial fracture-pin surgery11. Another particularly useful behavioral paradigm is the hot plate assay, which traditionally measures the latency to paw withdrawal to a thermal stimulus. While this assay has been used for decades12, truly a gold standard in preclinical pain research, measuring reflexive behavior alone has its limitations13. As a result, we have developed a modified hot plate paradigm that can capture elements of both reflexive and nonreflexive responses in the setting of a thermal stimulus14.
This modified hot plate assay determines the initial response latency as in the original hot plate test and an extended observation period to record additional nocifensive behaviors. By categorizing these extended behaviors into distinct categories (flinching, licking, guarding, jumping), the nonreflexive response to the thermal stimulus can be captured. Flinching is the rapid removal of the paw and/or splaying of digits, but the limb is quickly returned to the hot plate. Licking and biting of the hind and front paws are both defined as licking for analysis. Guarding is the continued raising of the limb beyond when afferent nociceptive information ends. Finally, jumping is the removal of all four limbs from the hot plate surface. These behaviors can be analyzed individually and grouped together with special care to still note the initial response latency.
All methods used while conducting this research were performed in compliance and with approval by the Stanford University Administrative Panel on Laboratory Animal Care (APLAC #33114) in accordance with American Veterinary Medical Association guidelines and the International Association for the Study of Pain. Mice (C57BL/6J, 9-11 weeks old upon arrival, 11-12 weeks old at study initiation) were housed 2-5 per cage and maintained on a 12 h light/dark cycle in a temperature-controlled environment with ad libitum access to food and water. Male mice weighed approximately 25 g at the start of the study. See the Table of Materials for details regarding all materials used in this study.
1. Baseline behavior measurements
2. Anesthesia/preparation
3. Surgery
4. After surgery
5. Hot plate testing
NOTE: Postinjury measurements can begin 7 days after tibial fracture-pin surgery. To avoid the effect of learning in this paradigm, perform the test once after surgery and compare to uninjured controls.
The tibial fracture-pin orthotrauma model reproduces the bone, muscle, and pain-like behaviors seen in complex human injury. As shown in Figure 1C, the tibial fracture heals over time, forming a callus at the fracture site that is still seen at 4 weeks post injury. As a result of the lateral approach with the bone saw described above (step 3.5), the tibialis anterior muscle is injured, becoming extensively fibrotic, as seen by increased collagen deposition throughout the tissue (Figure 1D). Hind paw sensitivity is quite profound, as evidenced by decreased threshold to mechanical stimuli, lasting at least 5 weeks after injury11. In addition, we have incorporated novel non-reflexive assays to more fully explore pain-like behaviors after peripheral injury14,15. As shown in Figure 3, orthotrauma injury results in increased reflexive flinching and non-reflexive behaviors (licking, guarding). Note that in some cases, mice also exhibit jumping, which is considered a non-reflexive behavior.
Figure 1: The orthopedic injury model involving unilateral tibial fracture and resulting in muscle fibrosis. (A) Schematic of the location of the tibia and tibialis anterior muscle, which are both injured in this model. (B) Intraoperative photograph demonstrating location of tibial bone fracture (black arrow), osteotomy and intramedullary nail (white arrow), and TA muscle. (C) microCT scan of the right tibia without fracture (left) or 4 weeks after fracture (right), demonstrating clear callus formation. (D) Orthotrauma results in extensive fibrosis of the TA muscle, as demonstrated by immunohistochemical staining of TA muscle sections showing increased collagen (white) and altered muscle fiber pattern (laminin, green) as well as loss of regular, central nuclei (DAPI, blue). Scale bars = 2 mm (C), 100 μm (D). This figure was adapted, with permission, from Tawfik et al. 202011. Abbreviations: TA = tibialis anterior; microCT = Micro Computer Tomography; DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Figure 2: Set-up for download and use of NCH Prism Software. (A) This panel shows a screenshot of the options that are available after opening NCH Prism Software. To utilize the free version, click on Continue to Use the Demo Version. (B) Once the program is fully open, a new window will open (shown here in panel B) where all the videos can be uploaded. Click on the green plus sign to add video files or drag files into the window. Once the videos are uploaded in the NCH Prism, double-click on a single video to open a large viewing window. (C) An example of a video being viewed in the NCH Prism is shown here. Note the millisecond timestamp at the bottom-right corner of the window and the cursor that can be dragged manually through the length of the video. Please click here to view a larger version of this figure.
Figure 3: Raster plots showing distinct nocifensive behaviors in an orthotrauma (injured) animal two weeks after injury compared to an uninjured control using a hot plate assay. (A) Raster plot showing flinching, guarding, and licking behaviors in a control animal. (B) Raster plot showing increased flinching, guarding, and licking behaviors in an injured animal. Please click here to view a larger version of this figure.
Critical steps within the protocol
It is crucial to maintain sterile conditions throughout the surgery. Moreover, proper animal care before, during, and after the surgery is paramount for the successful generation of the model. As mentioned earlier in the protocol, when performing the surgery, fracture the bone from the lateral side to ensure muscle injury. Take care not to fracture the tibia too low (below the advised junction between the middle and distal thirds of the tibia) because this will affect how the bone fractures, the researcher's ability to fix it with a pin, and the healing of the fracture.
To further ensure the translatability of preclinical findings to clinically useful treatments, the tibial fracture-pin model must be combined with outcome measures that have clear human correlates. Using the modified hot plate assay described above allows for the observance of temperature sensitivity due to chronic pain models in rodents. Individuals with chronic pathological conditions often report increased sensitivity and pain with changes in temperature. Increased temperature can lead to increased inflammation and impact the way that tissues expand and contract. Therefore, all preclinical researchers are encouraged to consider these approaches in designing their studies to evaluate acute and chronic postsurgical pain. One important part of obtaining reliable and reproducible results from the modified hot plate assay is to follow a strict protocol for determining individual behaviors on review of the videos. We have a very protocolized regimen for training new researchers on the video review and require them to meet the desired metrics for interrate reliability prior to having them score videos independently.
Limitations, modifications, and troubleshooting of the technique
Since the protocol described includes the use of a 27 G needle as an implant, one has to modify it to pair it with certain kinds of imaging, which may be adversely affected by a metal implant, for example, computed tomography (CT) or magnetic resonance imaging (MRI). Therefore, for imaging applications, replacing the metal implant with a suitable ceramic or plastic implant is advisable.
We have previously performed the hot plate assay at 52.5 °C and recorded significant differences in latency and nonreflexive behaviors, which were increased after peripheral injury15 and suppressed by morphine14. That said, we have opted to perform these hot plate experiments at a lower temperature of 50 °C going forward to better discriminate more subtle findings that may arise from less severe injuries or less potent analgesics. It is therefore recommended that those adopting this new technique trial multiple temperatures ensure that they do not run into a ceiling effect in their particular model.
Significance with respect to existing methods
Orthopedic injuries involving bone, muscle, and nerve trauma are a major public health concern common in many settings, including falls, sports-related trauma, motor vehicle accidents, and military combat11. Preclinical researchers need mouse models of orthopedic trauma that mimic multi-tissue injury, including bone, muscle, and nerve, since they are most representative of human orthopedic trauma. The tibial fracture-pin model was created to meet this need. Indeed, it has been shown that this model reproduces many features of orthopedic trauma seen in humans11.
Any future applications of the technique
In addition to studying mechanisms of trauma-induced pain, the fracture-pin model can also be used to evaluate drug candidates for orthopedic pain treatment. It can also be used to study fracture healing and evaluate the effects of different candidate therapeutics for fracture healing.
The authors have nothing to disclose.
GM is supported by an NDSEG Graduate Fellowship and a Stanford Bio-X Honorary Graduate Fellowship. VLT is supported by NIH NIGMS grant #GM137906 and the Rita Allen Foundation.
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Buprenorphine | Fidelis Pharmaceuticals | https://ethiqaxr.com/ordering/ | |
Ceramic implant (alternative to pin) | RISystem | RIS.221.103 | https://risystem.com/platefixation/mousescrew |
Chux (Absorbent Underpad) | Fisher Scientific | NC0059881 | https://www.fishersci.com/shop/products/underpad-17×24-chux-300-cs/nc0059881#?keyword=true |
C57BL/6J mice | The Jackson Laboratory | Jax #00664 | https://www.jax.org/strain/000664 |
Cotton swabs | Uline | S-18991 | https://www.uline.com/Product/Detail/S-18991/First-Aid/Cotton-Tipped-Applicators-Industrial-6 |
Cutting pliers | Amazon | B076XYVS6Y | https://www.amazon.com/iExcell-Diagonal-Cutting-Nippers-Chrome-Vanadium/dp/B076XYVS6Y |
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Drill bits | Amazon | B00HVIGSX2 | https://www.amazon.com/Universal-Diamond-Dremel-Rotary-Tool/dp/B00HVIGSX2 |
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High definition video camera | The Imaging Source | DFK 22AUC03 | https://www.theimagingsource.com/products/industrial-cameras/usb-2.0-color/dfk22auc03/?adsdyn&gclid=Cj0KCQiA3-yQBhD3ARIsAHuHT64uIIlImBvh_ toh-3GFSgBcL_fRc1gQTDyXlqDEa Qu4n2_VbWEiRuIaAiueEALw_wcB |
Inhalational anesthesia system | Kent Scientific | https://www.kentscientific.com/products/vaporizer-with-vetflo-single-channel-anesthesia-stand/ | |
Iodine solution | Amazon | B005FR7XIK | https://www.amazon.com/Dynarex-Povidone-Iodine-Scrub-Solution/dp/B005FR7XIK |
Iodine swab sticks | Amazon | B001V9QKMG | https://www.amazon.com/POVIDONE-IODINE-SWAB-1202-25Box/dp/B001V9QKMG |
Isoflurane | California pet pharmacy | https://www.californiapetpharmacy.com/fluriso-isoflurane-250ml.html | |
NCH Prism Software | https://www.nchsoftware.com/prism/index.html | ||
Plastic Cylinder | Amazon | B08R5KM5B6 | https://www.amazon.com/FixtureDisplays-Acrylic-Diameter-Thickness-15140-8-NPF/dp/B08R5KM5B6 |
Saline | Fisher Scientific | NC9054335 | https://www.fishersci.com/shop/products/saline-injection-0-9-10ml/NC9054335 |
Scalpel | Fisher Scientific | 12-000-162 | https://www.fishersci.com/shop/products/high-precision-10-style-scalpel-blade/12000162#?keyword= |
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Thermal place preference apparatus | BIOSEB | BIO-T2CT | https://www.bioseb.com/en/pain-thermal-allodynia-hyperalgesia/897-thermal-place-preference-2-temperatures-choice-nociception-test.html |
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